Use of prolines for improving growth and other properties of plants and algae

ABSTRACT

Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.09/493,039 filed Jan. 27, 2000.

STATEMENT REGARDING FEDERAL RIGHTS

This invention was made with government support under Contract No.W-7405-ENG-36 awarded by the U.S. Department of Energy to The Regents ofThe University of California. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates generally to growth of plants and algaeand, more particularly to the use of the chemical class of compoundsknown as prolines for improving the properties and performance of plantsand algae.

BACKGROUND OF THE INVENTION

Many agricultural activities are time sensitive, with costs and returnsbeing dependent upon rapid turnover-of crops or upon being first toreach the market place. Therefore, rapid plant growth is an economicallyimportant goal for many agricultural businesses that grow high-valuecrops such as vegetables, berries, and bananas, as well as for thegreenhouse and nursery businesses. The importance of improved cropproduction technologies has increased as a result of the observationthat yields for many well-developed crops have tended to plateau inrecent years. The goal of rapid plant growth has been addressed innumerous studies of plant regulatory mechanisms, which remainincompletely understood. In particular, a complete understanding has notbeen attained for the plant regulatory mechanisms that coordinate carbonand nitrogen metabolism, which must have a major impact on plant growthand development.

The metabolism of carbon and nitrogen in photosynthetic organisms mustbe regulated in a coordinated manner to assure efficient use of plantresources and energy. Understanding of carbon and nitrogen metabolismnow includes details of certain steps and metabolic pathways which aresubsystems of larger systems. In photosynthetic organisms, carbonmetabolism begins with CO₂ fixation which includes two major processestermed C-3 and C-4 metabolism. In plants with C-3 metabolism the enzyme,ribulose bisphosphate carboxylase (RuBisCo) catalyzes the combination ofCO₂ with ribulose bisphosphate to produce 3-phosphoglycerate, a threecarbon compound (C-3), that the plant uses to synthesizecarbon-containing compounds. In plants with C-4 metabolism, CO₂ iscombined with phosphoenol pyruvate to form acids containing four carbons(C-4) in a reaction catalyzed by the enzyme phosphoenol pyruvatecarboxylase. The acids are transferred to the bundle sheath cells wherethey are decarboxylated to release the CO₂ which is then combined withribulose bisphosphate in the same reaction as employed by C-3 plants. Inphotosynthetic organisms, nitrogen is assimilated by the action of theenzyme glutamine synthetase which catalyzes the combination of ammoniawith glutamate to form glutamine.

Previous research focusing on important enzymes and the genes encodingfor them has investigated the enzyme catalyzing the assimilation ofnitrogen to form glutamine. One such study is “Modulation of GlutamineSynthetase Gene Expression In Tobacco by the Introduction of an AlfalfaGlutamine Synthetase Gene in Sense and Antisense Orientation: Molecularand Biochemical Analysis” by Stephen J. Temple et. al., Mol. Gen. Genet236, 315 (1993), the teachings of which are hereby incorporated byreference herein. Therein it is stated that plants overexpressing theglutamine synthetase (GS) gene were visibly greener than control plants.Although, GS1-overexpressing plants exhibited about 45% increase intotal soluble protein and the GS1 antisense expressing plants exhibitedabout 40% decrease in total soluble protein, no mention was made of morerapid growth rate or greater plant yields. In “Overexpression of aSoybean Gene Encoding Cytosolic Glutamine Synthetase in Shoots ofTransgenic Lotus coroniculatus L. Plant Triggers Changes in AmmoniumAssimilation and Plant Development” by Remi Vincent et. al., Planta 201,424 (1997), it is stated that a 50% to 80% increase in total leaf GSactivity in transgenic plants is followed by degradation of shootprotein and early floral development. As these properties arecharacteristic of senescent plants, Vincent et al. states that the overexpression of GS in shoots may accelerate plant development, thusleading to premature flowering and early senescence. Again, no mentionis made,of any increase in plant yield or rate of growth. In“Overproduction of Alfalfa Glutamine Synthetase in Transgenic TobaccoPlants” by Peter Eckes et. al., Mol. Gen. Genet. 217, 263 (1989), theauthors state that GS overproducing plants were fertile and grewnormally, and that a high level of expression of a key metabolic enzymesuch as glutamine synthetase does not interfere with growth andfertility of plants. No mention was made of the ratio of leaf-to-root GSactivity, which has subsequently been measured by the present inventorsto be approximately equal to that for wildtype plants although theactivities in both the leaves and the roots have increased.

In “Does Root Glutamine Synthetase Control Plant Biomass Production inLotus Japonicus L.?” by Anis Limami et al., Planta 209, 495 (1999), theauthors show that over expression of root GS activity depresses biomassproduction. Two transgenic lines that were observed to grow poorly wereinvestigated and the difference in growth between these lines iscorrelated with the amount of root GS activity; the poorest growth wasresponsive to the highest GS root activity. Leaf GS activity wasobserved to remain constant in the plants. Limami et al. do not describea method for increasing biomass production or increasing plant growthrate or plant dry mass.

Numerous studies of nitrogen metabolism have found that variousmetabolites are important in the plants regulation of nitrogenmetabolism. These compounds include the organic acid malate and theamino acids glutamate and glutamine. Two previous patents have beenissued in this general area. In U.S. Pat. No. 5,840,656, for “Method forIncreasing Fertilizer Efficiency” which issued to Alan Kinnersley et.al., on Nov. 24, 1998, an improved fertilizer composition is describedthat includes conventional fertilizers and an amino acid mixture whichincludes glutamic acid. U.S. Pat. No. 5,739,082 for “Method of Improvingthe Yield of Herbicide-Resistant Crop Plants” which issued to GunterDonn on Apr. 14, 1998, reports the unexpected improvement in the yieldof transgenic crop plants which have been engineered to be resistant toGS inhibitors resulting from the treatment of these plants with very lowlevels of GS inhibitors. The compound glufosinate [glufosinate-ammonium(ammonium DL-monalanin-4-yl-methyl phosphinate)] acts as a GS inhibitorbecause it is a structural analog of the GS substrate, glutamic acid. GSis responsible for the detoxification of NH₃, and when GS is inhibitedby the glufosinate, the plant is severely damaged or destroyed by thetoxic accumulation of NH₃. No mention is made of the ratio ofleaf-to-root GS activity.

The compound, 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) issynthesized and metabolized in plants by the sequential action oftransaminase and hydrolyase enzymes. Similar transaminase and hydrolyaseenzymes and the metabolite, 2-hydroxy-5-oxoproline, have been identifiedin animal livers and kidneys. These enzymes and the2-hydroxy-5-oxoproline were partially characterized as described in “TheGlutamine Transaminase-ω-Amidase Pathway” by Arthur L. Cooper and. AltonMeister, CRC Critical Reviews in Biochemistry, pages 281-303 (January1977), and “Enzymatic Preparation of α-Keto Acids” by Alton Meister, J.Biochem. 197, 304 (1952). However, no physiological function wasattributed to these compositions in animals.

Accordingly, it is an object of the present invention to improve theproperties of plants, including growth rate, fresh weight and dryweight, by increasing the effective quantity of compositions therein topositively affect these properties.

Additional objects, advantages and novel features of the invention willbe set forth, in part, in the description that follows, and, in part,will become apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

SUMMARY OF THE INVENTION

To achieve the foregoing and other objects of the present invention, andin accordance with its purposes, as embodied and broadly describedherein, the composition hereof includes prolines in an amount effectiveto increase the rate of growth of plants and improve other properties ofgrowing plants.

In another aspect of the present invention in accordance with itsobjects and purposes the method for increasing the rate of growth ofplants and for improving other properties of growing plants hereofincludes contacting a plant with an effective amount of a proline.

It is preferred that the proline is applied to the foliar portion of theplant.

Preferably, a solution of 2-hydroxy-5-oxoproline (2-oxoglutaramate) isapplied to the foliar portion of the plant.

Preferably also 2-hydroxy-5-oxoproline is applied at a rate sufficientto maintain an effective concentration of the proline in the leaf duringthe growing period of the plant.

Benefits and advantages of the present invention include significantimprovement in plant properties, such as growth rate, nodulation, freshweight and dry weight, with a simple and efficient plant treatmentregimen. Similar effects have been observe for algae.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form, a part ofthe specification, illustrate the embodiments of the present inventionand, together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1a is a brief schematic representation of the biosynthesis of2-hydroxy-5-oxoproline in the nitrogen assimilation process, and itsrelationship to carbon metabolism in a typical plant, while FIG. 1b is asimplified schematic of the regulatory function of the2-hydroxy-5-oxoproline in the carbon metabolism and nitrogenassimilation processes in a typical plant in order to illustrate thechanges in the foliar concentration thereof affects plant growth.

FIG. 2 shows the fresh weight of plants as a function of the ratio ofthe leaf/root_((wildtype)) glutamine synthetase activity for wild typeand four transgenic lines (lines 1,3,6, and 8) of tobacco, andillustrates that not all of the transgenic plant lines have the desiredproperties.

FIG. 3 is a graph showing the foliar concentration of2-hydroxy-5-oxoproline (thick lines) in oats (x's) treated with thiscompound and in transgenic tobacco plants (open circles) and the freshweights (FWT) of these plants (thin lines), plotted as a function of theratio of their leaf/root_((wildtype)) GS activity.

FIG. 4 is a graph showing the emergence of the 3^(rd) and 4^(th) leavesfor switchgrass treated with 2-hydroxy-5-oxoproline, versus that foruntreated switchgrass plants, as a function of time.

DETAILED DESCRIPTION

Briefly, the present invention includes increasing the concentration ofprolines in the foliar tissues of a plant to improve plant growth andother properties. This can be accomplished in four ways: (1) theapplication of a solution of the proline directly to the foliar portionsof the plant by spraying these portions; (2) applying a solution of theproline to the plant roots; (3) genetically engineering the plant andscreening to produce lines that over-express glutamine synthetase in theleaves which gives rise to increased concentration of the metabolite,2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate);and (4) impairing the glutamine synthetase activity in the plant rootswhich causes increased glutamine synthetase activity in the leaves whichgives rise to increased concentration of 2-hydroxy-5-oxoproline.

Glutamine synthetase activity is a measure of the effectiveness of GS asa catalyst in the synthesis of glutamine and, ultimately, in thesynthesis of 2-hydroxy-5-oxoproline. The present invention has beenshown to raise the concentration of the 2-hydroxy-5-oxoproline in thefoliar portions of treated plants which, as a consequence, have beenobserved to have better growth properties than untreated plants. Thatis, the treated plants exhibit greater fresh weight and percent dryweights and demonstrate an up-regulated carbon dioxide fixation ratewith associated carbon metabolism, and a greater quantity ofphotosynthetically derived chemical energy than untreated plants as aresult of the greater ratio of dry weight to fresh weight. The inventioncan be used to increase the economic value and return of agriculturalcrops. Additionally, prolines can be used as growth enhancers forgreenhouse and other non-field growth environments. It should bementioned at this point that not ali plants of a chosen species areresponders. It may be necessary to select plants which respond toincreased foliar 2-hydroxy-5-oxoproline concentrations by observingtheir growth or other characteristics. Self-fertilized offspring ofresponding plants breed true. Prolines have been found to have a similareffect on algae.

Reference will now be made in detail to the preferred embodiments of thepresent invention, examples of which are illustrated in the accompanyingFigures. Coordination of carbon and nitrogen metabolism requires amechanism for monitoring the two metabolic systems. The metabolite,2-hydroxy-5-oxoproline has been identified in this monitoring process.It is believed by the present inventors that 2-hydroxy-5-oxoproline ismade in a futile cycle in the plant. Futile cycles are short pathwayswith only a few steps that generate unusual and critical metabolites.Turning now to FIG. 1a hereof, it may be observed that this metaboliteacts as a monitor of carbon or nitrogen flux through the main metabolicpathway. Thus, changes in the concentration of the2-hydroxy-5-oxoproline are transmitted through signaling pathways andultimately trigger changes in gene expression and consequently changesin metabolism. These characteristics of futile cycles are reflected inthe creation and destruction of this metabolite and in the significantchanges that occur in plants treated with exogenous2-hydroxy-5-oxoproline as will be demonstrated hereinbelow.

Also shown in FIG. 1a hereof, plants catalyze the conversion ofglutamine to synthesize 2-hydroxy-5-oxoproline through the action of atransaminase enzyme. Thus, the concentration of 2-hydroxy-5-oxoprolineis linked to the concentration of glutamine in the plant, assuming therate of destruction of this compound through a hydrolyase activity whichremoves the amide nitrogen of the 2-hydroxy-5-oxoproline and converts itto α-ketoglutarate, is approximately constant. The2-hydroxy-5-oxoproline has been shown to be present in plant leaves andthe synthetic activity of the transaminase enzyme is capable ofgenerating the observed concentrations of 2-hydroxy-5-oxoproline.

FIG. 1b shows schematically that since the concentration of2-hydroxy-5-oxoproline is linked to the concentration of glutamine,increased glutamine concentration results in the generation ofadditional 2-hydroxy-5-oxoproline which promotes growth bysimultaneously coordinating carbon fixation and metabolism and nitrogenassimilation,and metabolism. In order for this process to result inplant growth, an increase in the concentration of 2-hydroxy-5-oxoprolinein the leaves is required. A number of progeny of plants which overexpress the GS gene (transgenic) have been found to grow rapidly. Theseplants are observed to contain an increased ratio of leaf-to-root GSactivity, and an increased GS activity in the leaves which results in anincreased concentration of 2-hydroxy-5-oxoproline in the leaves. Anotherway to accomplish the improved growth is by direct foliar treatment ofthe plant with 2-hydroxy-5-oxoproline, or by sufficient quantities ofthis metabolite being applied to the plant roots to allow fortranslocation to the leaves of the effective quantity.

It will be shown that when plants are grown in the presence of2-hydroxy-5-oxoproline, the compound is taken up through the roots andtransported to the leaves. Alternatively, plants may be treated byapplication of a solution of 2-hydroxy-5-oxoproline to the foliarportions thereof. Since prolines are readily degraded by commonlyoccurring microbes, foliar treatment is expected to be more economicaland efficient. Treated plants have been found to contain elevated levelsof the compound. Oats, alfalfa, tomato, cantaloupe, cotton and lettuce,all C-3 plants; and switchgrass, a C-4 plant, were chosen asrepresentatives of the major plant groups based on their carbon-fixationmetabolism, and were treated with 2-hydroxy-5oxoproline. Overall plantgrowth and other growth-related indicia such as plant fresh weight andplant dry weight were monitored.

The immediate effects of increases in plant foliar2-hydroxy-5-oxoproline concentrations may be monitored by observingincreased CO₂ fixation rates. Plants having increased CO₂ fixation rateswere found to exhibit increased growth rates, while as the level of2-hydroxy-5-oxoproline diminishes, these functions were also found todiminish. Effective treatment dose is species dependent, and may also bedetermined by monitoring the growth of the treated plants, for example,by visual inspection or weighing the plants, and comparing these resultswith those for untreated plants. Effective treatment doses have tendedto be lower for foliar application, because increasing the concentrationin the foliar tissues has been observed to be essential to the plantresponse. Single treatments and periodic treatments during plant growthhave been undertaken. The treatment frequency or schedule employeddepends upon the plant species and upon the goals of the user. Possiblegoals include faster growth during a specific phase of the plant's lifesuch as early growth in a greenhouse to allow for faster transplantingof plants while requiring less time in the greenhouse, helping toovercome transplant shock for plants, or faster growth sustained in thefield, nursery, or greenhouse. A single treatment was sufficient toproduce increased growth rate in cantaloupe and cotton seedlings.

Transgenic plant lines genetically engineered to over express GS intheir foliar portions (while GS in the root portions were normal orimpaired) were found to produce increased 2-hydroxy-5-oxoproline levelsin their foliar portions, which was found to lead to markedly increasedgrowth rates over wildtypes; root GS activity is approximately that ofthe wildtype (unaltered) plants for these plants. FIG. 2 shows the ratioof the leaf/root glutamine synthetase activity for wildtype and for fourtransgenic tobacco plant lines, and illustrates that not all of thetransgenic plant lines have the desired properties. The transgenicplants were allowed to self-fertilize (selfing) to permit geneticsegregation and produce offspring having increased numbers of copies ofthe glutamine synthetase transgene to increase expression of GS activityin the leaf tissues while allowing essentially normal or impaired GSactivity in the root tissues of the same plants. These are the plantsthat exhibit increased growth rate and improved plant properties, whilethose plants which have been genetically engineered to over express GSand which have not been screened and genetically segregated by selfingor cross pollinating do not exhibit an increased leaf-to-root GSactivity ratio and do not present improved properties. Therefore, plantsmust be selected for increased growth and genetically segregated byselfing as an example with continued selection until a line breeds true(genetically homozygous for the desired trait), so that the trait isexpressed in all offspring in subsequent generations.

FIG. 3 is a graph illustrating the foliar concentration of2-hydroxy-5-oxoproline expressed as a percentage of that in controlplants (thick lines) for oats (x's) treated with 2-hydroxy-5-oxoprolineand for transgenic tobacco plants (open circles), and the fresh weightpercent for the treated oat plants and transgenic tobacco plantsexpressed as a percentage of that for control plants (thin lines), bothas a function of leaf/root ratio of GS activity. The root GS activity isthat of the wild-type (unaltered) plants. It may be observed thatincreased leaf/root GS concentration ratios yield greater concentrationsof 2-hydroxy-5-oxoproline in the leaves and greater fresh weights.

A dramatic increase in growth rate of algae growing photosyntheticallyhas been observed when treated with 2-hydroxy-5-oxoproline. Continuouslyculturing the algae in the presence of this compound or mixtures of thiscompound with other prolines will enrich sub-strains of the algae thatrespond well to the prolines.

Having generally described the invention, the following EXAMPLES provideadditional details of the effects of the metabolite,2-hydroxy-5-oxoproline and other prolines on plant properties. In theEXAMPLES, the concentration of 2-hydroxy-5-oxoproline in plant leaveswas determined by extracting the 2-hydroxy-5-oxoproline into acidifiedwater from a known weight of fresh leaf tissue. The sampled leaves werefully extended and photosynthetically active. The amount of2-hydroxy-5-oxoproline was measured using a high-performance liquidchromatograph fitted with an optical detector (210 nm) and a columnpacked with a sulfonated polystyrene di-vinyl benzene polymer suitablefor ion-exclusion chromatography of Kreb cycle intermediates. Themetabolite was eluted with 0.01N H₂SO₄ in water. The chromatograph peakcorresponding to 2-hydroxy-5-oxoproline was identified by the knowncolumn retention time of 2-hydroxy-5-oxoproline. A standard curve wasprepared using chemically synthesized 2-hydroxy-5-oxoproline (structureconfirmed using 13-C nuclear magnetic resonance) and varying the amountof this standard injected into the system. The area integrated under thepeak was correlated with the known amount of standard injected into thechromatograph. The amount of 2-hydroxy-5-oxoproline in leaf tissuesamples could then be quantitatively determined from the area under theappropriate peak.

Glutamine synthetase activity was measured using the transferase assayas described in “Glutamine Synthetase (E. coli)” by B. Shapiro and E.Stadtman, Meth. Enzym. 17A, 910 (1970). The GS activity is expressed inmicromoles per gram of fresh weight of tissue per minute.

Also in the EXAMPLES, plants were grown at 73° F. with 16 h length daysand 8 h length nights. Fluorescent lights were supplemented withincandescent bulbs to generate intensities of 1760, 980 or 360microeinsteins per meter squared per second as indicated in theEXAMPLES. Plants were grown in a standard potting mixture of peat moss,vermiculite and perlite, and nutrients were supplied by watering dailywith the commonly used Hoagland's plant nutrient solution which containsan array of known plant nutrients and is considered to be appropriate tomeet the nutrient needs of plants as they normally receive them fromsoil.

Transgenic lines were created as described in Temple et al., supra. Theplants in each of these lines were allowed to self-fertilize in at leastthe T-0, and T-1, generations. Self fertilization was performed topermit genetic segregation to produce offspring with increased numbersof copies of the glutamine synthetase transgene to increase expressionof glutamine synthetase activity in the leaf tissues and to allow forother molecular or genetic events to either maintain essentially normalor impaired glutamine synthetase activity in the root tissues of thesame plants. Therefore, plants must be selected for increased growth andreproduced by selfing with continued selection until a line breeds true.This screen for increased growth was done among and within the varioustransgene lines. The seeds of each of the generations were germinated onMurashige and Skoog medium containing kanamycin. The presence ofkanamycin in the medium permitted the identification of and thescreening of the transgenic plants resulting from each generation. Theseeds from the T-1 generation, the T-2 generation, were thus germinatedand grown to young plants. The ratio of leaf/root glutamine synthetasewas then determined by sampling the leaves and roots of these T-2generation plants and measuring the glutamine synthetase activities.

In the practice of the present invention, plants were treated regularlyin order to sustain increased CO₂ fixation and increased metabolic ratesby directly measuring the CO₂ fixation rate repeating treatment whenthis rate declines. Multiple treatments over several weeks were found togive the best plant growth, development, and the most improved relatedphysiological parameters.

The concentrations of 2-hydroxy-5-oxoproline set forth in Tables 1, 2,5, and 6, hereinbelow, show the concentrations of this material in theplant leaves.

Turning now to the EXAMPLES:

EXAMPLE 1 Daily Root Treatment of Oats (C-3 Plant) with2-hydroxy-5-oxoproline

TABLE 1 shows the results of harvesting oat plants (C-3 plant)(commercial cultivar Lodi) whose roots were treated daily with2-hydroxy-5-oxoproline. The treatment was commenced when the seedlingswere 7 days old and continued for 56 days; the 2-hydroxy-5-oxoprolinewas supplied daily to their roots as 50 ml of between 0.5 to 50 μMsolutions of 2-hydroxy-5-oxoproline in Hoagland's plant nutrientsolution having a pH of 6.3. Oats treated with 2-hydroxy-5-oxoprolinecontained higher percent dry weight than did controls. When the amountof dry weight was expressed as a function of fresh weight, the percentdry weight of the treated plants ranged from 8.1 to 8.4% when comparedwith 7.6% for untreated control plants.

TABLE 1 2-Hydroxy-5-oxoproline Parameter/ Whole Plant Concentration inLeaf Plant Fresh Weight (FWT), g (micromoles/gFWT) Control 14.95 4.9Treated 18.06 7.0

EXAMPLE 2 Weekly Foliar Treatment of Lettuce (C-3 Plant) with2-hydroxy-5-oxoproline

TABLE 2 shows the results for lettuce (a C-3 plant) where treatment wasinitiated with 7 -day old plants. The fresh weight, dry weight, percentdry weight, 2-hydroxy-5-oxoproline concentration in the leaves, and theCO₂ fixation rates are all increased in the treated plants. The greaterdry weight percent increases the value of these plants and that of algaeas fuel because the increase provides a greater amount of combustiblematerial per gram of fresh weight (that is, a greater amount of carbonwas fixed by the plant), and because an increase in dry weight percentreduces the water content in the fuel which is an energy cost when thefuel is combusted. It also increases the value of these plants assources of biomass suitable for conversion to other products such aschemicals, liquid fuels, or hydrogen, to name a few examples. Treatmentswere applied weekly to the foliar parts of the plants using an air brushuntil approximately 20% drip. The aqueous treatment solution contained2-hydroxy-5-oxoproline (10 mg/liter) in 0.07% sodium laurel sulfate, and1.2% glycerol at a pH of 6.3. The plants were harvested at 63 days ofage after 7 treatments; the harvest was made 7 days after the lasttreatment. These plants were provided Hoagland's plant nutrient solutionto their roots and grown under light intensity of 380 microeinsteins.

TABLE 2 2-Hydroxy-5- Whole Plant oxoproline CO₂ Fixation Param- FreshWhole Plant Concentration in Rate (micro- eter/ Weight Dry Weight Leaf(micro- moles CO₂ Plant (FWT) g (DW) g moles/gFWT) fixed/m²/sec) Control55.3[100%] 4.00[100%]  6.25[100%] 6.6^(a), 6.9^(b) Treated 65.2[118%]6.35[159%] 13.37[212%] 9.0^(a), 9.6^(b) ^(a)34 day-old and ^(b)57day-old plants

^(a) 34 day old and ^(b) 57 day-old plants

At harvest (63 days of age) none of the 12 control plants had emergedflowering structures, whereas 6 of the 12 treated plants hadwell-developed flowering structures.

EXAMPLE 3 Daily Root Treatment of Alfalfa (C-3 Plant) with2-hydroxy-5-oxoproline

TABLE 3 shows the results of treatment of alfalfa plants (a C-3 plant)with 2-hydroxy-5-oxoproline. Growth and nodulation were improved by thetreatment. The alfalfa commercial cultivar Saranac, was grown asdescribed hereinabove using 1760 microeinsteins per meter squared persecond as the light intensity, and watered daily with quarter-strengthHoagland's plant nutrient solution that had been modified to replace thenitrogen (nitrate) with chloride in the solution. Nodulating alfalfaseedlings were treated beginning at about 4 days of age, with the samequantity of 2-hydroxy-5-oxoproline as described in EXAMPLE 1 for plantsreceiving the 2-hydroxy-5-oxoproline through their roots.

TABLE 3 Whole plant Nodule number/ Nodule weight/ Plants Fresh weight(%) plant (%) plant (%) Controls 100 100 100 Treated(soil) 145 214 165

The time to flowering or emergence of seed structures of treated dailyand untreated plants was tracked in alfalfa and oats. The treatedalfalfa plants flowered 2-5 days sooner than did the untreated plants,while the treated oat plants displayed seed structures 3-5 days earlierthan did the untreated control plants.

EXAMPLE 4 Daily Root Treatment of Switchgrass (C-4 plant) with2-hydroxy-5-oxoproline

Switchgrass (a plant with C-4 metabolism, commercial cultivar, Alamo)plants were treated with 2-hydroxy-5-oxoproline and when harvested hadgrown better than had controls and as such treated plants had greaterfresh weights than controls and also had a higher content of dry weightwhen expressed per unit of fresh weight. Average fresh weight of thefoliar part of the plants was 115% of that for the control plants. Thedry weight was increased in these plants (16.5% and 12.05% in foliarparts and roots, respectively) in such a manner to provide a greateramount of fixed carbon per unit of fresh weight than untreated plants(14.9% and 16.7% in foliar parts and roots, respectively. The plantswere treated as described in EXAMPLE 1 and grown as described in thepreceding section under 1760 microeinsteins per meter squared per secondof light intensity, and the plants were harvested and the fresh weightsmeasured. The better growth of the treated plants is furtherdemonstrated by tracking the emergence of specific leaves in treated andcontrol plants as shown in FIG. 4 which shows the treated switchgrassplants consistently emerged leaves sooner than did the control plants.

EXAMPLE 5 Daily Root Treatment of Oats (C-3 Plant) with2-hydroxy-5-oxoproline; Selection of Responders

TABLE 4 shows the results of screening a population of wildtype oats(commercial cultivar Lodi) for those plants which respond to treatmentwith 2-hydroxy-5-oxoproline applied through their roots. Oats weretreated with 2-hydroxy-5-oxoproline through their roots as described inEXAMPLE 1. Individual plants that responded to the increased level of2-hydroxy-5-oxoproline were identified by measuring the rate of CO₂fixation using a CO₂ fixation meter at the maximum light intensity forphotosynthetic carbon dioxide fixation of 2000 microeinsteins per metersquared per second. The rates were measured in the leaves of treatedplants on two successive days and the plants were allowed to mature andproduce seed. Other parameters of rapid growth such as number of tillersor rate of leaf emergence were also useful in choosing a population ofplants that had a greater fraction of members which respond to2-hydroxy-5-oxoproline treatment. The progeny of this selection werefound to maintain the response trait.

TABLE 4 CO₂ fixation Rate as Number of Individual Differential CO₂Tillers in Plant Concentration, ppm Mature Plant Treated Plants 28.9 13Responders 28.0 18 25.9 17 25.3 17 25.2 21 24.5 19 24.2 12 24.2 15 23.621 23.2 17 Average of Responders 25.3 17.0 Non-Responders 19.9 14 19.613 17.2 15 16.1 14 Average of Non- 18.2 14.0 Responders Untreated Plants18.9 14 16.9 14 18.9 13 Average of Untreated 18.2 13.7 Plants

EXAMPLE 6 Daily Root Treatment of Oats (C-3 Plant) with2-hydroxy-5-oxoproline; Effects of Increasing Leaf/root GS Ratio

TABLE 5 and FIG. 3 hereof show the results of increasing the ratio ofleaf/root GS activity on plant growth and on the concentration of2-hydroxy-5-oxoproline in the leaves of oat plants treated with acompound (tabtoxinine-β-lactam) that binds to glutamine synthetase andimpairs the normal destruction of this enzyme, thereby causing the leafGS activity to increase while the root GS activity decreases. (Formethodology, see, e.g., T. Knight, D. Bush, and P. Langston-Unkefer,Plant Physiol. 88, 333 (1988)). The compound (Tabtoxinine-β-lactam) wasdelivered to the roots. The plants were found to grow rapidly andoutgrew oats not treated. When allowed to grow without treatment, theoats grow at normal rates and were observed to contain the same ratio ofleaf/root GS ratio that is found in the wildtype oats. The treated,rapidly growing oats were observed to have sharply increased GS activityin their leaves and diminished glutamine synthetase in the roots; thusthese plants had an increased leaf/root GS ratio. The level of2-hydroxy-5-oxoproline was also observed to increase in the leaves ofthese plants as a consequence of the increased leaf/root GS activityratio.

TABLE 5 Leaf/ Root 2-Hydroxy-5- Whole Plant GS Activity GS oxoproline inTreatment Fresh Weight (micromoles/g Activity Leaf (micro- Of Oats (FWT)(g) FWT/min) Ratio moles/g FWT) Controls 14.95 Leaf: 10.5 1.35 4.9 Root:7.8 Tabtoxinine- 24.6 Leaf: 25.9 6.6 16.6 β-lactam- Root: 3.9 treatedoats 2-Hydroxy- 18.07 Leaf: 16.9 2.64 7.04 5-oxoproline Root: 6.4treated Oats

EXAMPLE 7 Genetically Increasing the Leaf/root GS Ratio in TobaccoPlants

TABLE 6 shows the results of genetically increasing the ratio ofleaf/root glutamine synthetase activity on plant growth and level of2-hydroxy-5-oxoproline in the plants. Transgenic lines of tobaccocontaining an increased ratio of leaf/root glutamine synthetase activitywere screened for in the T-2 generation. The screening was done bymeasuring the activity of glutamine synthetase in the leaf and foottissues of plants and determining the ratio of leaf/root glutaminesynthetase activity. Plants having increased leaf/root GS ratio grew atfaster rates than did their counterparts with the normal ratio ofleaf/root GS ratio as shown in TABLE 6. See also FIG. 2 hereof.

TABLE 6 Leaf/ Root 2-Hydroxy-5- Whole Plant GS Activity GS oxoproline inPlant Fresh Weight (micromoles/g Activity Leaf (micro- Treatment (FWT)(g) FWT/min) Ratio moles/g FWT) Control 21.8 Leaf: 24.0 3.9 6.0 Root:6.3 Line 1 46.8 Leaf: 43.1 8.6 16.1 Root: 5.1 Line 8 36.1 Leaf: 32.6 6.99.7 Root: 4.7

EXAMPLE 8 Effects of 2-hydroxy-5-oxoproline on Algae

The growth rate of algae, Chlorella sorokinian (obtained from theAmerican Type Culture Collection), was increased when2-hydroxy-5-oxoproline was added to the growth medium. The massaccumulation growth curve for this single-cell photosynthetic organismis described as a sigmoidal function. The time it takes to reach themid-log phase of growth was substantially reduced for treated algae(approximately 40 h) when compared with untreated algae (approximately60 h). This reflects substantially accelerated growth for the treatedalgae cultures when compared with untreated cultures. The growth mediumfor algae growth is described in Biotechnology & Bioengineering Vol. IVby Daboll et al., pages 281-297 (1962). Carbon dioxide was provided asthe sole carbon source and was allowed to sparge into the cultures ofboth treated and untreated cultures. The light source suppliedapproximately 980 ft candles. Media pH was initially adjusted to 8.0using either NaOH or HCl, and subsequently maintained at pH 7.0 bycontrolling the introduction of CO₂.

EXAMPLE 9 Foliar Treatment of Oats (C-3 Plant) with 5-oxoproline(2-pyrrolidone-5-carboxylic acid)

Foliar treatment of oat plants with the structural analog of2-hydroxy-5-oxoproline, 5-oxoproline (2-pyrrolidone-5-carboxylic acid)also resulted in increased plant growth rate. The treated plants werefound to yield fresh foliar weights that were 126% of the foliar weightsfor untreated control plants when harvested 17 days after the singletreatment. A solution containing 40.5 mg per milliliter of2-pyrrolidone-5-carboxylic acid and 1% dimethylsulfoxide in water at apH of 6.3 was applied, but other solutions of this compound were alsofound to be effective. These plants were grown under lights of 980microeinsteins per meter squared per second and the treatment wasapplied to their foliar parts.

EXAMPLE 10 Daily Root Treatment of Alfalfa (C-3 Plant) with2-hydroxy-5-oxoproline; Effect on Insect Infestation

Plants having increased 2-hydroxy-5-oxoproline concentration were foundto have fewer insect pests infesting them than did the control plants.Thrip infestation of plants having increased 2-hydroxy-5-oxoprolineconcentration was compared with that of control plants having a normallevel of 2-hydroxy-5-oxoproline in their leaves. Control (80 plants) andplants with elevated levels of 2-hydroxy-5-oxoproline (80 plants) weresubjected to thrip infestation; these two sets of plants were randomlyintermingled in the greenhouse to assure equal exposure to the pest. Ofthe set of control plants, 15 were infested with thrip while only 11 ofthe other set of plants were infested with thrip one week after initialexposure to the pests. The light intensity for growth was approximately1760 microeinsteins per meter squared per second.

EXAMPLE 11 Single or Multiple Foliar Treatment of Other Plants with2-hydroxy-5-oxoproline

TABLE 7 and TABLE 8 show the results when commercial vegetables such ascantaloupe, and tomatoes, and a crop such as cotton were tested for theeffects of the metabolite on their growth rates. These plants receivedone foliar application of 2-hydroxy-5-oxoproline as young seedlings.These plants grew more rapidly as determined by either fresh weight orleaf length. The cantaloupe and cotton seedlings were harvested andtheir fresh weight was determined.

TABLE 7 Foliar Fresh Weight, g Foliar Fresh Weight, g Plant and VarietyUntreated plants Treated plants Cantaloupe-Imperial 0.85 1.55*Cotton-Pima 1.27 1.85* Tomato-Halley 3155 11.97** 19.08** *Plants were16 days old and were harvested 9 day after the treated plants hadreceived a single treatment. **Plants were 24 days old and treatedplants had received 5 weekly treatments.

*Plants were 16 days old and were harvested 9 day after the treatedplants had received a single treatment.

** Plants were 24 days old and treated plants had received 5 weeklytreatments.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching. For example, plants may be treated with prolines byinjection of these materials into plant stems, pseudo stems or trunks.Moreover, because increasing the leaf concentration of2-hydroxy-5-oxoproline has been shown to lead to improved growth andother plant properties, other genetic methods might be employed forincreasing the foliar concentration of 2-hydroxy-5-oxoproline; amongthem: (1) the over expression of the transaminase gene followed byscreening for improvements in plant properties; and (2) impairment ofthe amidase gene which assists in the removal of 2-hydroxy-5-oxoprolinefrom the cells, followed by screening for improvements in plantproperties.

The embodiments were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed is:
 1. A method for increasing the rate of growth ofalgae over that for wild type algae, comprising contacting algae with aneffective amount of a proline selected from the group consisting of2-hydroxy-5-oxoproline (2-oxoglutaramate) 5-oxoproline(2-pyrrolidone-5-carboxylic acid and mixtures thereof).